Construction of a gamete-enriched gene pool and RNAi-mediated functional analysis in Dictyostelium discoideum

Muramoto, Tetsuya; Suzuki, Katsuya; Shimizu, Hajime; Kohara, Yuji; Kohriki, Eiko; Obara, Shinji; Tanaka, Yoshimasa; Urushihara, Hideko

doi:10.1016/s0925-4773(03)00136-9

Cited by 32 publications

(45 citation statements)

References 30 publications

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“…This would suggest that MEGAP1 may not Journal of Cell Science 121 (7) be a functional activator of GTPases and that MEGAP2, -3 and -4 could be functional activators of GTPases. MEGAP1 and MEGAP2 were chosen initially, as both are available from the Japanese cDNA project (Muramoto et al, 2003) and thus demonstrably expressed. We chose to apply the MEGAP nomenclature, as we can find no evidence of an association with SCAR/WAVE in D. discoideum (see below).…”

Section: Resultsmentioning

confidence: 99%

“…discoideum homologues of MEGAP were identified by BLAST searches against the Tsukuba Dictyostelium cDNA database (Muramoto et al, 2003) and the Dictyostelium genome project libraries (Eichinger et al, 2005) using the FCH domain of mammalian MEGAP. mgp1 (DictyBaseID DDB0188795) was cloned from cDNA (AHN288) into the HindIII/NotI sites of PCR Blunt TOPOII vector (Invitrogen).…”

Section: Knockout Constructsmentioning

confidence: 99%

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Dictyostelium MEGAPs: F-BAR domain proteins that regulate motility and membrane tubulation in contractile vacuoles

Heath

Insall

2008

Journal of Cell Science

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PCH family proteins are fundamentally important proteins, linking membrane curvature events with cytoskeletal reorganisation. One group, the MEGAPs (also called srGAPs and WRPs) contain RhoGAP domains in addition to the F-BAR domain. We disrupted MEGAP1 and MEGAP2 in Dictyostelium both singly and in combination. We found a strong cytoskeletal phenotype in MEGAP1– cells and a subtle phototaxis defect in MEGAP2– slugs. MEGAP1–/2– cells have an overabundance of filopodia and slug motility and function are affected. The most dramatic changes, however, are on contractile vacuoles. MEGAP1–/2– cells empty their contractile vacuoles less efficiently than normal and consequently have three times the usual number. GFP-tagged MEGAP1 localises to tubules of the contractile vacuole network and when vacuoles start to empty they recruit cytosolic GFP-MEGAP1. Mutants in the Saccharomyces homologues RGD1 and RGD2 also show abnormal vacuoles, implying that this role is conserved. Thus, MEGAP is an important regulator of the contractile vacuole network, and we propose that tubulation of the contractile vacuole by MEGAP1 represents a novel mechanism for driving vacuole emptying.

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Section: Resultsmentioning

confidence: 99%

Section: Knockout Constructsmentioning

confidence: 99%

Dictyostelium MEGAPs: F-BAR domain proteins that regulate motility and membrane tubulation in contractile vacuoles

Heath

Insall

2008

Journal of Cell Science

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“…The organisation of the Dictyostelium mating-type locus does not closely resemble previously studied sex-determining regions (supporting online text S5), although like them the mat locus must ultimately control a transcriptional cascade (21). Whether the key genes directly regulate transcription or through downstream targets remains to be determined.…”

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confidence: 88%

“…S6). MatD is distantly related to the Hap2-GCS1 family of gamete fusion proteins (20), though another Dictyostelium discoideum gene, hapA, encodes a protein much more closely related to the canonical Hap2 group, and is enriched in cells competent for mating (21).…”

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confidence: 99%

Sex Determination in the Social AmoebaDictyostelium discoideum

Bloomfield

Skelton

Ivens

et al. 2010

Science

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The genetics of sex determination remains mysterious in many organisms including some that are otherwise well-studied. Here we report the discovery and analysis of the mating-type locus of the model organism Dictyostelium discoideum. Three forms of a single genetic locus specifies this species' three mating-types: two versions of the locus are entirely different in sequence, and the third resembles a composite of the other two. Single, unrelated genes are sufficient to determine two of the mating-types, while homologues of both these genes are required in the composite type. The key genes encode polypeptides that possess no recognisable similarity to established protein families. Sex determination in the social amoebae thus appears to use regulators unrelated to any currently known.Most eukaryotes are sexual, but little is known in molecular detail about sex across most branches of the eukaryotic tree. One aspect, the genetic basis of sex determination, is well understood in several animal, fungal and plant lineages (1-5), but across the protozoan kingdoms we know little, and nothing in comparable detail. The social amoebae are members of the Amoebozoa, and have an unusual sexual cycle that leads to the formation of dormant, walled macrocysts (6) ( Fig. 1 A and B). To produce a macrocyst, a pair of haploid amoebae of different sexes fuse (7) to form a diploid zygote, which then attracts surrounding haploid cells (8). These help to lay down external layers of cellulose around the developing mass of cells before being cannibalized by the zygote (9). After a period of dormancy the cyst germinates, releasing haploid progeny that arise most likely after meiosis and multiple mitoses (10). Population genetics of wild isolates indicate that mating and recombination are probably frequent in the wild (11).The most-studied species of social amoeba, Dictyostelium discoideum, is notable for having three sexes (hereafter called mating-types I, II, and III; supporting online text, S1), as well as uncommon self-fertile homothallic strains (12)(13)(14). Each of the three sexes can pair with each of the other two, but not with itself, giving three possible classes of zygote: type-I/type-II, type-I/type-III, and type-II/type-III. Although several genes are known to be involved during the sexual cycle (15), the determinant of mating-type has proved elusive. Genetic analysis suggested that mating-type is stable, and determined by a single locus with two or * To whom correspondence should be addressed. garethb@mrc-lmb.cam.ac.uk Phone: +44 (0) (10,14,16). We argued that it might be possible to identify this postulated locus by searching for genes which are present in any member of one mating-type but absent (or highly diverged) in any member of another. For this purpose we performed comparative genomic hybridizations using DNA microarrays composed of probes for around 8500 of the 10500 predicted genes in the sequenced type-I D. discoideum genome (17).We analysed ten strains derived from independent wild isolates (table S1) usi...

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“…It also did not affect the sexual potency of the cells, leaving the actual gene for gp138 unknown. Genes expressed specifically in the gamete stage were screened by constructing a gamete-enriched gene pool (23). Although certain genes involved in the signal transduction pathway, such as ras and rac, were highly elevated in the gametes, their functions are unknown.…”

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confidence: 99%

A Surface Glycoprotein Indispensable for Gamete Fusion in the Social Amoeba Dictyostelium discoideum

et al. 2012

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bSexual reproduction is essential for the maintenance of species in a wide variety of multicellular organisms, and even unicellular organisms that normally proliferate asexually possess a sexual cycle because of its contribution to increased genetic diversity. Information concerning the molecules involved in fertilization is accumulating for many species of the metazoan, plant, and fungal lineages, and the evolutionary consideration of sexual reproduction systems is now an interesting issue. Macrocyst formation in the social amoeba Dictyostelium discoideum is a sexual process in which cells become sexually mature under dark and submerged conditions and fuse with complementary mating-type cells. In the present study, we isolated D. discoideum insertional mutants defective in sexual cell fusion and identified the relevant gene, macA, which encodes a highly glycosylated, 2,041-amino-acid membrane protein (MacA). Although its overall similarity is restricted to proteins of unknown function within dictyostelids, it contains LamGL and discoidin domains, which are implicated in cell adhesion. The growth and development of macA-null mutants were indistinguishable from those of the parental strain. The overexpression of macA using the V18 promoter in a macA-null mutant completely restored its sexual defects. Although the macA gene encoded exactly the same protein in a complementary mating-type strain, it was expressed at a much lower level. These results suggest that MacA is indispensable for gamete interactions in D. discoideum, probably via cell adhesion. There is a possibility that it is controlled in a mating-typedependent manner.

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Construction of a gamete-enriched gene pool and RNAi-mediated functional analysis in Dictyostelium discoideum

Cited by 32 publications

References 30 publications

Dictyostelium MEGAPs: F-BAR domain proteins that regulate motility and membrane tubulation in contractile vacuoles

Dictyostelium MEGAPs: F-BAR domain proteins that regulate motility and membrane tubulation in contractile vacuoles

Sex Determination in the Social AmoebaDictyostelium discoideum

A Surface Glycoprotein Indispensable for Gamete Fusion in the Social Amoeba Dictyostelium discoideum

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